Uses of Modified RNA Encoding Retinaldehyde Dehydrogenase

This application claims priority under 35 U.S.C. § 119(e) to U.S. provisional application number, U.S. Ser. No. 62/743,943, filed Oct. 10, 2018, which is incorporated herein by reference.

The origin and development of vaccines was a turning point in human history and the permanent fight against microbes. Today, vaccines are the most cost-effective way to save lives. Most current vaccines are administered by injection through the skin, which often results in weak or no immune protection at mucosal sites. Because the major point of entry for many human pathogens occurs at gastrointestinal (e.g., polio virus,, HIV-1), respiratory (e.g. influenza virus,, adenovirus), or genital (, HIV-1, HPV) mucosal surfaces, protective immunity against mucosal pathogens requires the development of vaccine strategies capable of inducing mucosal immune responses (1, 2). For instance, diarrheal diseases constitute the second leading cause of death (after pneumonia) in young children of developing countries (3), and the design of tailored vaccines to the pathogen(s) and site of infection constitute a great challenge.

Priming of adaptive immune responses, particularly by dendritic cells (DCs), in specific mucosal sites determines subsequent homing of antigen-specific T and B cells to the mucosal source tissue, as well as to other mucosal tissues (4, 5). Therefore, in order to target the intestinal mucosa, most vaccine formulations are designed for oral administration. However, oral vaccines require high dosages of antigen to induce an immune response due to poor antigen stability in the harsh conditions of the gastrointestinal tract, and because a tolerogenic, rather than immunogenic, response often results from oral antigen exposure (6). Moreover, despite the efficacy of oral vaccines in developed countries, their efficacy in developing countries is often unsatisfactory. Nutritional status, ongoing persistent infections with helminths and other parasites, and the intestinal microbiota are thought to play a major role in the vaccines' differential efficiency (7).

Likewise, vaccines targeting memory responses in the uterine or vaginal mucosa require the generation of tissue resident memory cells (5). In such cases, uterine vaccination strategies may generate local immunity, but clinical translation has been found challenging (8).

Thus, there is a need to develop robust parenteral vaccine formulations that are efficiently targeted to mucosal tissues and possess mucosal imprinting properties.

Provided herein are modified polynucleotides encoding retinaldehyde dehydrogenase (RALDH). RALDH was selected due to its ability to indirectly upregulate mucosal homing receptors on activated lymphocytes, resulting in a targeted antigen-specific response. Therefore, the modified polynucleotides may be used, for example, as components of vaccines. As described herein, vaccines comprising modified polynucleotides encoding RALDH may be used, for example, to target mucosal tissues, leading to the generation of antigen-specific immunity in said tissues. In one embodiment, the modified polynucleotides, e.g., mRNA, may comprise at least one pseudouridine in place of a uridine and/or at least one 5-methylcytosine in place of a cytosine. Other modifications, as described herein, are also possible. The modified polynucleotides can form the basis of new parenteral vaccine formulations that target mucosal tissues, bypassing the tolerogenic effects commonly associated with mucosal vaccinations as well as the toxicity mediated by other molecules, which also have mucosal imprinting properties, such as all-trans retinoic acid (ATRA). The vaccine, which can, in some embodiments, induce mucosal receptors on antigen-specific T cells, may be used in cancer immunotherapy, particularly in regard to tumors developing at mucosal surfaces.

Provided herein are novel modified polynucleotides (e.g., mRNA) encoding a RALDH protein. The invention, in some aspects, includes a variety of vaccines comprising the modified polynucleotides (e.g, mRNA) described herein. The vaccines may be used, for example, to treat infections (e.g., mucosal infections) or cancers (e.g., mucosal cancers). In some instances, the modified polynucleotide may be used in a tolerogenic vaccine. Another aspect of the invention provides a kit comprising the modified polynucleotide and/or the vaccine described herein.

The modified polynucleotides (e.g., mRNA), in some embodiments, comprise an open reading frame (ORF) encoding a retinaldehyde dehydrogenase (RALDH) protein (e.g., human RALDH), wherein at least one uridine is pseudouridine, and/or at least one cytosine is 5-methylcytosine. Other modifications, as described herein, are also contemplated.

In some embodiments, the RALDH protein is selected from the group consisting of: retinaldehyde dehydrogenase 1 (RALDH1) protein, retinaldehyde dehydrogenase 2 (RALDH2) protein, and retinaldehyde dehydrogenase 3 (RALDH3) protein. In one embodiment, the RALDH protein is RALDH2. In an embodiment, the RALDH protein is a human RALDH protein or variant thereof. Exemplary amino acid and nucleotide sequences of RALDH isoforms are provided herein.

In some embodiments, the open reading frame encodes two RALDH proteins selected from the group consisting of: retinaldehyde dehydrogenase 1 (RALDH1) protein, retinaldehyde dehydrogenase 2 (RALDH2) protein, and retinaldehyde dehydrogenase 3 (RALDH3) protein. In another embodiment, the open reading frame encodes retinaldehyde dehydrogenase 1 (RALDH1) protein, retinaldehyde dehydrogenase 2 (RALDH2) protein, and retinaldehyde dehydrogenase 3 (RALDH3) protein.

In another aspect, the invention provides a vaccine comprising at least one antigen, and at least one modified ribonucleic acid (e.g., mRNA) polynucleotide comprising an open reading frame (ORF) encoding a retinaldehyde dehydrogenase (RALDH) protein.

In some embodiments, the vaccine described herein further comprises an adjuvant (e.g., alum, AS03, AS04, MF59, or TLR agonists). In other embodiments, the vaccine described herein further comprises retinal, retinol, β-carotene, or a combination thereof.

The antigen may be, for example, a polynucleotide, protein, peptide, plasmid, virus, viral fragment, bacteria, bacterial fragment, fungi, fungal fragments and conjugate.

In some embodiments, the vaccine described herein is a mucosal vaccine. In such vaccines, the antigen may be, for example, a viral or a bacterial pathogen, or a combination thereof.

In other embodiments, the vaccine may be a cancer vaccine, and the antigen may be a tumor antigen, e.g., a mucosal tumor antigen. Exemplary mucosal tumor antigens include guanylyl cyclase C, sucrose isomaltase, CDX1, CDX2, mammoglobulin, small breast epithelial mucin, RAGE antigen, MUC1, and neoantigens. In some embodiments, the mucosal tumor antigen is associated with a mucosal cancer selected from the group consisting of colon cancers, head and neck squamous cell carcinomas, lung cancers, cervical cancers, and pancreatic cancers.

The vaccine may be formulated in a variety of different ways, for example, as a nanoparticle, microparticle, liposome, or hydrogel. In one particular example, the vaccine is formulated as a cationic lipid nanoparticle. The vaccine may further comprise a pharmaceutically acceptable excipient.

In yet another aspect, the invention provides a tolerogenic vaccine. Tolerogenic vaccines are used, for example, to reduce an immune response (induce tolerance) in the face of a pathological or unwanted activation of the normal immune response, which occurs, for example in autoimmune disorders. The tolerogenic vaccine, in one aspect, comprises an antigen, an immunomodulatory agent, and at least one modified messenger ribonucleic acid (mRNA) polynucleotide comprising an open reading frame (ORF) encoding a retinaldehyde dehydrogenase (RALDH) protein. Exemplary immunomodulatory agents include, but are not limited to, mTOR inhibitors, HDAC inhibitors, MHC-peptide complexes, and antigen-laden erythrocytes.

The vaccines or pharmaceutical compositions thereof described herein may be used in methods to induce antigen-specific immune responses (e.g., a T cell response or a B cell response), for example, in the mucosal tissues of a subject. Depending on the content of the vaccine of pharmaceutical composition thereof, it may be used to immunize a subject against a pathogen (e.g., a mucosal pathogen). The vaccines or pharmaceutical compositions thereof may also be used to treat an infection (e.g., a mucosal infection) or a cancer (e.g., a mucosal cancer) in a subject.

The vaccines or pharmaceutical compositions thereof may be administered parenterally, for example, by subcutaneous administration or intramuscular administration, or orally. The vaccine or pharmaceutical composition thereof may be administered as a single dose, or as a single dose followed by one or more subsequent booster doses.

Another aspect of the invention provides a kit comprising: a polynucleotide described herein; a pharmaceutically acceptable excipient; a container; and instructions for using the kit. Further kits comprising any one of the vaccines or pharmaceutical compositions thereof are also described herein.

The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, the Drawings, the Examples, and the Claims.

As used herein and in the claims, the singular forms “a,” “an,” and “the” include the singular and the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to “an agent” includes a single agent and a plurality of such agents.

The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, RNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5′ to 3′ direction unless otherwise indicated.

The term “modified polynucleotide” (e.g., modified RNA, “modRNA”), as used herein, refers to a polynucleotide (e.g., DNA, RNA) that comprises at least one modified nucleotide. For example, the polynucleotide may comprise any of the nucleoside analogs, chemically modified bases, biologically modified bases, intercalated bases, modified sugars, isomers, and/or modified phosphate groups described herein.

The term “open reading frame” (ORF), as used herein, refers to a continuous stretch of RNA beginning with a start codon (e.g., AUG) and ending with a stop codon (e.g., UAA, UAG, UGA) that encodes a protein. In some embodiments, the protein encoded by the ORF is a retinaldehyde dehydrogenase (RALDH) protein.

In some embodiments, the ORF is codon-optimized. As used herein, “codon-optimized polynucleotide” refers to a polynucleotide that comprises codons that do not match those of the wild-type polynucleotide, but that do not alter the translated amino acid sequence of the encoded protein. The optimized codons can be used, for example, to increase mRNA stability, reduce secondary structures, minimize tandem repeat codons or base runs (which may impair gene construction or expression), manipulate transcriptional and translational control regions, add or delete protein trafficking sequences, insert, delete, or shuffle protein domains, add or delete restriction sites, or match codon frequencies in target and host organisms (for proper folding and secondary structure). Codon optimization tools, algorithms and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods.

The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. A protein may comprise different domains, for example, a nucleic acid binding domain. In some embodiments, a protein is in a complex with, or is in association with, a nucleic acid, e.g., RNA. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook,(4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.

In some embodiments, the protein is a RALDH protein. There are three splice variants (isoforms) of RALDH, termed RALDH1, RALDH2, and RALDH3. RALDH proteins are enzymes that catalyze the synthesis of retinoic acid (RA) from retinaldehyde, β-carotene, and vitamin A (retinol). The amino acid sequences of each isoform are given below:

The term “untranslated region” (UTR), as used herein, refers to a series of nucleic acids which are transcribed but not translated. Each polynucleotide generally has a UTR flanking each terminus of the ORF: a 5′ UTR which begins at the transcription start site and continues to the start codon but does not include the start codon, and a 3′ UTR, which begins immediately following the stop codon and continues until the transcriptional termination signal.

The term “recombinant” as used herein in the context of proteins or nucleic acids refers to proteins or nucleic acids that do not occur in nature, but are the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations as compared to any naturally occurring sequence.

The term “vaccine,” as used herein, refers to one or more agents administered to a subject in order to stimulate the production of antibodies and provide immunity against one or more diseases.

The term “effective amount” or “therapeutically effective amount,” as used herein, refers to an amount of a biologically active agent (e.g., a vaccine) that is sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of a vaccine comprising modified RNA encoding RALDH may refer to the amount of vaccine necessary to treat a given disease or disorder, e.g., to generate a therapeutically effective antigen-specific immune response in the subject. As will be appreciated by the skilled artisan, the effective amount of an agent, e.g., a vaccine, may vary depending on various factors as, for example, on the desired biological response, e.g., on the specific antigen used, disease targeted, and on the modified RNA being used.

The terms “administer,” “administering,” or “administration,” as used herein refers to implanting, applying, absorbing, ingesting, injecting, or inhaling, the inventive polynucleotide (e.g., RNA), vaccine, or pharmaceutical composition thereof.

The term “subject,” as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development.

The terms “treatment,” “treat,” and “treating,” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. As used herein, the terms “treatment,” “treat,” and “treating” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

The present invention is based, at least in part, on the discovery that modified RNA encoding retinaldehyde dehydrogenase (RALDH) (modRNA-RALDH) can be used in vaccines to target immune response to mucosal tissues. As described herein, the use of modRNA-RALDH in parenteral vaccines leads to mucosal tissue homing, while avoiding exposure to toxic molecules, such as all-trans retinoic acid (ATRA). As a result, vaccines incorporating the modRNA-RALDH described herein are more efficacious than current mucosal tissue vaccines, which often have disagreeable tolerogenic effects. Many current mucosal vaccines require life-attenuated pathogens and/or potent adjuvants, resulting in suboptimal safety profiles. In contrast, the vaccines described herein are able to robustly target mucosal tissues without such side effects. It was also surprisingly found that the mucosal immune surveillance conveyed by ATRA, for example, resulting from administration of the vaccine formulations described herein, was not limited to only the small and large intestine, but extended to other mucosal surfaces, such as the oral cavity, nasal cavity, and urogenital tract. Accordingly, the modRNA-RALDH molecules provided herein may be formulated as pharmaceutical compositions and/or vaccines, and used to treat a number of diseases, including mucosal infections and cancers.

Some aspects of this disclosure provide modified mRNA (modRNA) encoding at least one RALDH enzyme (modRNA-RALDH). Without wishing to be bound by any particular theory, administration of the modRNA-RALDH results in the transient expression of RALDH, and, in the presence of vitamin A, generation of all-trans retinoic acid (ATRA) by antigen-presenting cells (e.g., dendritic cells). Antigen presentation by dendritic cells (DCs) exposed to modRNA-RALDH results in the upregulation of mucosal homing receptors on activated B and T cells. Thus, parenteral vaccine formulations containing modRNA-RALDH can generate antigen-specific immunity at mucosal tissues, without compromising the systemic immune surveillance, unlike other parenteral vaccines.

This mucosal immune surveillance is of relevance in the context of mucosal vaccination strategies targeting a host of mucosal pathogens, including, but not limited to,, enterotoxigenic, rotavirus,, and HIV-1. The use of modRNA-RALDH enhances the efficacy of parenterally (i.e., subcutaneously (SQ) or intra-muscularly (IM)) administered vaccines. Parenteral vaccines typically induce immune responses in skin-associated peripheral lymph nodes that drain the inoculation site and normally do not produce mucosa-tropic memory cells. By contrast, oral vaccination can naturally elicit mucosal memory, primarily focused on the small intestine, because APCs in gut-associated lymphoid tissues, unlike APCs in peripheral lymph nodes or the spleen, express RALDH and synthesize all-trans retinoic acid (ATRA). When lymphocytes are activated by antigen and simultaneously exposed to ATRA, they initiate a mucosa-homing program. However, vaccines applied to mucosal surfaces (e.g., orally or intranasally (IN)) are often poorly immunogenic and/or have suboptimal safety profiles due to the need of using either life attenuated pathogens or potent adjuvants that may exert toxic effects. It has been shown that the addition of soluble ATRA to vaccine formulations administered parenterally can induce a protective gut-homing memory response because after SQ injection, free ATRA enters local lymph vessels and is transported together with other vaccine components (e.g., antigen(s) plus adjuvant) to the draining lymph node. However, since free lymph-borne ATRA is not retained in lymph nodes, daily repeat injections over several (e.g., 5) days of relatively high doses of ATRA are needed to achieve mucosal imprinting. This is both impractical for clinical translation and poses a safety risk because high tissue concentrations of free ATRA can cause an inflammatory response at the injection site.

At the intestinal mucosa, dendritic cells (DCs) from gut-associated lymphoid structures (Peyer's patches (PP) and mesenteric lymph nodes (mLN)) induce the up-regulation of α4β7 integrin and chemokine receptor CCR9 on T and B lymphocytes (4, 9-11). As a result, these cells acquire the capacity to home to the small intestine. DCs originating from spleen or peripheral lymph nodes (pLN) cannot induce a similar gut-homing phenotype. This intestinal imprinting has been shown to result from the exclusive capacity of DCs from PP and mLN, but not pLN, to express retinal dehydrogenases (RALDH (9)), enzymes that convert dietary vitamin A to all-trans retinoic acid (ATRA (9)). Of note, the addition of exogenous ATRA to pLN DCs enables these cells to efficiently upregulate gut-homing receptors on activated lymphocytes. Experiments with human lymphocytes and DCs isolated from the mLN vs. spleen yielded analogous results (11). The exposure to ATRA during parenteral immunization was shown to convey protection againstinfections (12). Furthermore, it was found that mucosal immune surveillance conveyed by vaccine formulations containing ATRA is not limited to the small and large intestine, but parenteral ATRA exposure during immunization also generates antigen-specific T and B cells at other mucosal surfaces, such as the oral cavity (determined in the saliva), nasal cavity, and urogenital tract.

Accordingly, vaccines were designed to deliver RALDH enzymes to pLN DCs in order to generate ATRA capable of mucosal imprinting of antigen-specific B and T cells, which would confer a mucosal homing phenotype upon the impacted B and T cells. As demonstrated herein, the use of modRNA-RALDH in parenteral vaccines does allow for mucosal imprinting while avoiding tissue exposure to toxic ATRA; administration of modRNA-RALDH results in the transient expression of RALDH and, in the presence of vitamin A, generation of ATRA by antigen-presenting cells (e.g., DCs). Antigen presentation by DCs exposed to modRNA-RALDH results in the upregulation of mucosal homing receptors on activated B and T cells. Therefore, the vaccine formulations described herein generate antigen-specific immunity at mucosal tissues without compromising systemic immune surveillance, unlike classic parenteral vaccines. modRNA, in free form or packaged in cationic lipid nanoparticles, was found to have moderate adjuvant properties, act directly on cells with phagocytic capacity, and to be degraded intracellularly upon transient induction of protein expression. In addition, modRNA-RALDH can be readily administered either SQ or IM and can be combined with existing or new vaccine formulations to improve efficacy. Moreover, when studying the effect of ATRA on immune responses in skin-draining lymph nodes in vivo, it was found that mucosal memory responses were not only enhanced in the small intestine, but also at other mucosal surfaces, such as the female reproductive tract, the upper respiratory tract, and salivary glands. This finding was unexpected because earlier in vitro studies had suggested that the exposure of activated lymphocytes to ATRA selectively induces homing only to the small intestine, but not to other mucosal tissues. Thus, in addition to preventing intestinal infections, vaccine formulations that generate a sufficient amount of ATRA in peripheral lymph nodes may also have utility in preventing many other mucosal infections.

Hence, the modRNA-RALDH described herein allows for the design of robust parenteral vaccine formulations targeting mucosal tissues, bypassing the tolerogenic effects commonly associated with mucosal vaccinations, as well as toxicity mediated by other molecules with mucosal imprinting properties. Moreover, the induction of mucosal receptors on antigen-specific T cells by modRNA-RALDH means it may also be used in cancer immunotherapy, particularly in regard to tumors developing at mucosal surfaces.

Accordingly, the present invention is directed to modified polynucleotides encoding RALDH protein (modRNA-RALDH), as well as pharmaceutical compositions and vaccines comprising the modRNA-RALDH. The invention also includes methods of using the modRNA-RALDH, for example, to treat various diseases, including viral infections and/or cancers. A further aspect of the invention includes kits including the modRNA-RALDH.

Some aspects of this disclosure provide modified polynucleotides (e.g., modRNA) comprising an open reading frame (ORF) encoding a retinaldehyde dehydrogenase (RALDH) protein and functional fragments and variants thereof. In some embodiments, the RALDH is a human RALDH. In other embodiments, the RALDH is from a non-human source (e.g.,, and).

As discussed above, there are three isoforms of RALDH: RALDH1, RALDH2, and RALDH3. Provided below are three exemplary human RALDH ORF polynucleotide sequences:

In some embodiments, the ORF of the modRNA comprises a sequence 100% identical to any one of SEQ ID NOs: 1-3. In another embodiment, the ORF of the modRNA comprises a sequence that is 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to any one of SEQ ID NOs: 1-3.

In some embodiments, the ORF of the modRNA may encode RALDH1, RALDH2, or RALDH3. In one embodiment, the ORF of the modRNA encodes RALDH1. In one embodiment, the ORF of the modRNA encodes RALDH2. In one embodiment, the ORF of the modRNA encodes RALDH3. In another embodiment, the ORF of the modRNA encodes two of the following: RALDH1, RALDH2, and RALDH3. In other embodiments, the modRNA encodes RALDH1, RALDH2, and RALDH3.

Exemplary sequences of human RALDH1, RALDH2, and RALDH3 are presented herein as SEQ ID NOs: 4-6. In some embodiments, the ORF encodes a RALDH protein that is 100% identical to any one of SEQ ID NOs: 4-6. In other embodiments, the ORF encodes a RALDH protein that is 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to any one of SEQ ID NOs: 4-6.

RALDH typically comprises three domains: a NAD-binding domain (comprising a five-stranded parallel β-sheet), a catalytic domain (comprising a six-stranded parallel β-sheet), and an oligomerization domain (comprising a three-stranded anti-parallel β-sheet). In some embodiments, the ORF of the modRNA may encode one or more domains of RALDH1, RALDH2, and RALDH3, or a combination thereof. For example, the ORF may comprise a first domain from RALDH1, a second domain from RALDH2, and a third domain from RALDH3. In some embodiments, the ORF encodes two domains from a first RALDH protein and a single domain from a second RALDH protein.